4-Methylcytosine DNA modification is critical for global epigenetic regulation and virulence in the human pathogen Leptospira interrogans.
The main results reproduced: recomputed values matched the published ones within tolerance.
- ✓Any deviation was negligible
- 🟡Could not use the authors’ exact input data
- 🟡Reported values were only indirectly comparable
- 🟡A deviation arose in the data or preprocessing
- 🟡A deviation was attributed to the published material
- 🟡Reported values were not (fully) derivable from the shared data
- 🟡The central claim did not (fully) hold under reproduction
- 🟡Overall, the reproduction showed a material discrepancy
A 0–100 reproducibility-quality score from the per-question grades, shown as a z-score: standard deviations above (+) or below (−) the mean of comparable assessments.
▸Reproduction agent’s raw note
Described well enough to reproduce the RNA-seq pipeline numbers; mostly 1:1. The paper's headline computational results reproduce EXACTLY from the public GEO deposit (GSE138917): the 193 differentially expressed genes in the lomA mutant = the complementation-controlled set (m2011vsLP & m2011vsC2) - C2vsLP at padj<0.05; the 20 with >2-fold = those with |log2FC|>1 in both mutant comparisons; the 12,682 CTAG motifs = 6,341 palindromic sites x 2 strands. All three derivable from the deposit -> strong evidence the numbers are real (no fabrication). An independent raw re-run on «our HPC» (bowtie 1.2.2 -> featureCounts 2.0.6 -> pydeseq2 0.5.4) confirms in magnitude: counts reproduce the authors' deposited counts (log-r=0.976, 96.5% of cells within 10%), strict set = 215 (vs 193, +11%), 2-fold subset = 14 (vs 20); residual delta is the DE engine (pydeseq2 vs R DESeq2 1.20.0 - R unbuildable due to multi-room conda lock contention) + featureCounts version drift. NOT reproduced: the SMRT 4mC '80% of CTAG methylated' claim - the PacBio reads are deposited as basecalled subreads fastq with NO IPD kinetics, so modification re-calling is impossible from the deposit (data-insufficient, honest blocker). NOT attempted: Hi-C contact maps (PRJNA631225 + cited koszullab/E_coli_analysis pipeline present, but qualitative figure-level result, ~332M read pairs - out of cheap numeric scope) and all wet-lab assays (growth/adhesion/antibiotic/virulence, promoter mutagenesis). NOTE: the brief's Code link koszullab/E_coli_analysis is the koszul-lab Hi-C tool (E. coli) - it is genuinely cited by the paper for its Hi-C component (co-author Cockram), NOT for the RNA-seq/methylation results reproduced here. All grades provisional and human-checkable.
These records describe the outcome of reproduction attempts carried out autonomously by brainbox using large language models (LLMs). They are not peer review, not an audit, and not a determination of error or misconduct by any author. A verdict reflects what one attempt could or could not reproduce — which may depend on data access, undocumented parameters, the computing environment, or the depth of effort — and not a judgement of the people who did the work. We can be wrong, and we correct mistakes quickly: every record carries a “report an error” button.
Assessment versions
Every reproduction run is kept as an immutable version — anchored to the data as it stood, with a tamper-evident chain hash. A rerun (e.g. after an author updates a deposit) adds a new version; the previous one stays on record.
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v1 current initial assessment Score 78assessed: 2026-06-18 ⛓ ba81f53c6e0b
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Provenance — full disclosure
When this reproduction was carried out, which methodology version was used, and by whom — so the record can be audited and checked independently.
- Reproduced
- 2026-06-18
- Rubric version
- v1.0
- Assessed by
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🤖 AI curator · claude (ai-curator room) · v1.0 · run #1 2026-06-18no human curator yet
- Last updated
- 2026-08-05
Provisional, curator- or AI-assessed, and independently checkable. A reproduction outcome states what one attempt could reproduce — not a judgement of the authors.
Deep full-text extraction
Model: opusWhether and how an 'orphan' 4-methylcytosine (4mC) DNA methyltransferase controls key cellular processes and virulence in the understudied pathogen Leptospira interrogans.
- ★ A 4mC orphan MTase (lomA) is present only in the pathogenic (P) clade of Leptospira and methylates CTAG motifs. finding
- ★ Inactivation of the MTase abolishes CTAG motif methylation and causes genome-wide dysregulation of gene expression, acting as a global epigenetic regulator. mechanism
- ★ The MTase mutant shows growth defects, decreased adhesion to host cells, increased susceptibility to LPS-targeting antibiotics, and loss of virulence in an acute infection model. finding
- ★ Transcription of an ECF sigma factor (sigX) is altered in the methylase mutant and is directly controlled by methylation of CTAG motifs in its promoter and 5' coding region. mechanism
- ★ The regulon of the sigma factor is dysregulated in the methylase mutant and in a sigma-factor-overexpressing strain. finding
- ★ 4mC methylation is essential for the Leptospira bacterial life cycle and physiology, with implications for other understudied species. finding
- SMRT sequencing of a transposon insertion mutant verified the CTAG sequence motif targeted by the MTase. method
| Assay | System | Perturbation | Readout | Platform |
|---|---|---|---|---|
| SMRT (PacBio) genome sequencing and base modification/motif analysis | L. interrogans serovar Manilae (WT, lomA mutant, complemented) | MTase (lomA) transposon/insertion mutation and complementation | genome-wide methylated vs unmethylated CTAG motifs | Pacific Biosciences RS II; SMRTLink v6.0.0; Unicycler v0.4.7; EMBOSS v6.6.0.0 |
| bulk RNA-seq (rRNA-depleted, stranded) | L. interrogans exponential-phase cultures (WT, complemented, lomA) | MTase (lomA) mutation / complementation | differential gene expression | Illumina HiSeq 2500; Ribo-Zero; TruSeq Stranded mRNA; DESeq2 v1.20.0 |
| RT-qPCR | L. interrogans RNA samples | MTase mutation; CTAG motif mutation in sigX promoter | transcript levels normalized to flaB reference gene | BioRad CFX96 real-time system; SsoFast EvaGreen Supermix |
| Cell adhesion/translocation (qPCR-based) | Canine kidney epithelial MDCK-1 cells with L. interrogans | MTase mutation; none | bacterial abundance via flaB qPCR normalized to canine GAPDH | qPCR standard curve |
| Bacterial stress/antibiotic susceptibility (alamarBlue viability) | L. interrogans in EMJH | polymyxin B, colistin, H2O2, NaCl, lysozyme, penicillin G, Triton X-100 | viability/survival | alamarBlue reagent |
| In vivo virulence (acute infection) | Syrian Golden Hamsters (3-4 weeks) | intraperitoneal injection of WT vs mutant strains | survival/death, tissue bacterial load at 4 DPI | — |
| Chromosome conformation capture (3C/Hi-C) | L. interrogans mid-log cultures | none/strain comparison | genome-wide contact maps (5 kb bins) | Illumina TruSeq Nano; HpaII digestion; Bowtie2; SCN normalization |
| Immunoblotting (Western blot) and fluorescence (GFP) measurements | L. interrogans strains (FLAG-tagged Loa22; reporter constructs) | promoter swaps; CTAG/coding mutations | protein abundance (anti-FLAG, anti-FlaB loading control); GFP fluorescence | Li-Cor C-Digit scanner; Tecan Infinite M Nano spectrophotometer |
- ▼ Inactivation of the MTase resulted in complete abrogation of CTAG motif methylation.
- ▼ MTase mutants exhibited growth defects.
- ▼ MTase mutants showed decreased adhesion to host cells.
- ▲ MTase mutants had higher susceptibility to LPS-targeting antibiotics.
- ▼ MTase mutants were no longer virulent in an acute infection model.
- – Mutation of CTAG motifs in the sigX promoter altered its transcription.
- – The sigma factor regulon was dysregulated in the methylase mutant and in the sigma-factor overexpressing strain.
- – The orphan MTase is present only in the pathogenic P clade of Leptospira, not the saprophytic S clade.
- count over one million severe cases per year worldwide (global leptospirosis burden)
- count 60 000 fatalities per year (global leptospirosis deaths)
- pvalue adjusted P-value < 0.05 (threshold for differentially expressed genes (DESeq2, BH-corrected))
- pvalue FDR < 5% (COG enrichment threshold (Fisher test, BH-corrected))
- pvalue *P<0.05, **P<0.01, ***P<0.001 (significance thresholds for t-tests/ANOVA in figures)
- other P-value cutoff = 0.001; min methylation fraction = 0.3; min Qmod score = 30; 95% CI (SMRTLink base modification analysis parameters)
Statistical methods review
Model: sonnetA neutral, descriptive read of the statistical approach — what was done, and (for shared learning, not as criticism) what could also have been done.
The study used a three-strain comparative design (wild-type, lomA transposon mutant, and complemented L. interrogans) to characterize a 4mC methyltransferase. RNA-seq differential expression was assessed with DESeq2 (negative binomial generalized linear model) and Benjamini–Hochberg FDR correction; COG category enrichment was tested with Fisher's exact test, also BH-corrected. Phenotypic assays (growth, adhesion, stress, fluorescence) were compared using Student's t-tests or ANOVA with post-hoc corrections in GraphPad Prism. Results were reported with SEM error bars and asterisk-based significance thresholds rather than exact p-values.
| Test | Applied to | n | Assumptions |
|---|---|---|---|
| DESeq2 negative binomial generalized linear model with Wald test | Pairwise differential expression comparisons among WT, lomA mutant, and complemented strain (RNA-seq) | — | not stated |
| Fisher's exact test | COG category over- or under-representation among differentially expressed genes | — | not stated |
| Student's t-test (two-sample) | Phenotypic comparisons (growth curves, adhesion/translocation assays, fluorescence, stress assays) where two groups compared | — | not stated |
| ANOVA with post-hoc multiple comparisons (method not named; Prism default) | Phenotypic comparisons involving more than two groups or conditions | — | not stated |
| SMRT methylation detection p-value thresholding (SMRTLink Base Modification and Motif Analysis) | Genome-wide identification of methylated CTAG motifs | — | stated |
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Dispersion in phenotypic assay figures is reported as SEM↳ Could also: Standard deviation (SD) or 95% confidence intervals could also be used — SD describes the variability of the underlying measurements regardless of sample size; 95% CIs convey estimation precision directly; both are often preferred when biological replicate counts are small, because SEM decreases with n and can appear narrower than the biological variability warrants
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Statistical significance in phenotypic assays is communicated with asterisk thresholds (* < 0.05, ** < 0.01, *** < 0.001)↳ Could also: Exact p-values (e.g., p = 0.023) could also be reported for each comparison — Exact p-values allow readers to judge evidence strength directly and facilitate downstream meta-analyses or cross-study comparisons, whereas threshold asterisks convey only a categorical significance band
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RNA-seq differential expression was analyzed with DESeq2↳ Could also: edgeR (negative binomial GLM with TMM normalization) is a widely used alternative for the same data type — DESeq2 and edgeR implement related but distinct normalization and dispersion-estimation strategies; cross-validating key findings between the two is a common robustness check, particularly when biological replicate counts are small
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COG enrichment was assessed with Fisher's exact test applied independently to each category after BH correction↳ Could also: Gene Set Enrichment Analysis (GSEA) applied to the full ranked DESeq2 output could also be used — GSEA uses the continuous ranking of all genes rather than a binary differentially-expressed threshold, which can detect coordinated but modest signals across a functional category that a threshold-based overlap test may not capture
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ANOVA post-hoc multiple-comparison procedure is described only as Prism 'default parameters' without naming the specific method↳ Could also: Explicitly naming and reporting the post-hoc procedure (e.g., Tukey HSD for all pairwise comparisons, or Dunnett's test with WT as the reference) is also standard practice — Naming the procedure and its family-wise error rate control makes the multiplicity strategy reproducible and transparent, and allows readers to assess whether the chosen correction is appropriate for the comparison structure
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The number of independent biological replicates per condition is not stated for phenotypic assays↳ Could also: An explicit statement of biological replicate numbers, and optionally a power analysis or effect-size estimate informing sample size, could also be included — Reporting replicate counts allows readers to assess statistical power and is a standard expectation in quantitative phenotypic studies; it also supports reproducibility by others attempting to replicate the experiments
What was reproduced
The exact results taken into scope, with each reported value next to the value our attempt produced.
Scope — PMID 33301041
Paper: Gaultney, Vincent, Lorioux, Coppée, Sismeiro, Varet, Legendre, Cockram, Veyrier, Picardeau (2020). 4-Methylcytosine DNA modification is critical for global epigenetic regulation and virulence in the human pathogen Leptospira interrogans. Nucleic Acids Research 48(21):12102–12115. DOI 10.1093/nar/gkaa966. PMCID PMC7708080.
Datasets the paper relies on
- GSE138917 (SRP225811 / PRJNA577724) — RNA-seq, Illumina HiSeq 2500, 9 samples:
WT (LP1-3), complemented (C2-1..3), lomA mutant (m2011-1..3). GEO ships the
AUTHORS' processed DESeq2 tables (
*.complete.xls) + reference genome (CP011931.fa) + annotation (CP011931.gff). - PRJNA631225 — SMRT/PacBio reads for 4mC modification calling: SRR10257437 (WT), SRR10257436 (lomA), SRR10257435 (complemented).
Code link in brief = MIS-HARVEST (recorded, not a drop trigger)
The brief's Code: github.com/koszullab/E_coli_analysis is the Hi-C 3C analysis
repo for E. coli (Lioy/Cournac 2018 Cell). It contains NO Leptospira and is NOT
the RNA-seq/methylation pipeline of this paper. The link was harvested because
co-author C. Cockram is from the koszul lab. Per brief P16, we reproduce with the
described standard tools on the paper's own data — equally valid.
In scope (pipeline-derived, reproducible)
| id | reported result | paper loc | pipeline | path/cost |
|---|---|---|---|---|
| C1 | 193 genes significantly DE in lomA mutant vs WT (BH padj<0.05) | RNA-seq results / Methods | Sequana: Bowtie 1.2.2 → featureCounts 1.4.6-p3 → DESeq2 1.20.0 | verify vs shipped m2011vsLP.complete.xls; reproduce from SRA raw reads on «our HPC» |
| C2 | 20 genes with >2-fold difference (subset of the 193) | RNA-seq results | same | same table / raw reproduction |
| C3 | 12,682 CTAG motifs in the genome | Methods / results | motif count on CP011931.fa | trivial deterministic (cheap) |
| C4 | 80% of the 12,682 CTAG motifs methylated in WT | results | SMRT Base Modification & Motif Analysis (ipdSummary/SMRTLink), padj<0.001, methyl-frac≥0.3, Qmod≥30 | hard 20% — needs PacBio kinetics; feasibility TBD from SRA |
Out of scope (wet-lab / manual / not a pipeline)
- Growth defects, host-cell adhesion, antibiotic susceptibility, virulence in infection models (animal experiments).
- Promoter CTAG-motif mutagenesis of the ECF sigma factor (molecular cloning).
Strategy
- Tier A (cheap, on front1): pull shipped DESeq2 tables + genome; (i) verify 193 /
20 are derivable from the shipped
m2011vsLP.complete.xls(auditability / fabrication check); (ii) count CTAG motifs in CP011931.fa → expect 12,682. - Tier B («our HPC» SLURM): full raw reproduction — Bowtie→featureCounts→DESeq2 on the 9 SRA runs → independent DEG count vs 193.
- Tier C (hard 20%): SMRT 4mC calling if SRA reads carry kinetics.
Assessments & scoring basis
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Reproduction footprint
claude-opus-4-8Measured resources invested to assess this paper — sanitised (machine class only, no job ids/paths). Compute = HPC accounting (SLURM); tokens = the AI agent's session.